Diabetes, 59, 249-255, January, 2010

Hyperglycemia-Induced Reactive Oxygen Species Increase Expression of the Receptor for Advanced Glycation End Products (RAGE) and RAGE Ligands

高血糖により誘導される活性酸素種は RAGEと RAGEリガンドの発現を増加させる

Dachun Yao and Michael Brownlee

2015/01/05M1 瀧井靖歩

FIG. 1. Hyperglycemia-induced ROS increase expression of RAGE, S100 calgranulins, and HMGB1.A: Primary HAECs were infected with UCP1, SOD2, GLO1, or empty control adenovirus (CTL). After incubation with either low glucose (LG) (5 mmol/l) or high glucose (HG) (30 mmol/l) for 5 days, mRNA levels were determined by real-time PCR (n = 3). B: Intracellular protein levels of RAGE, S100A8, and HMGB1 were determined by Western blotting (n = 4). Protein sizes were evaluated by standard protein markers, and their sizes were as follows: RAGE (46 kDa), S100A8 (11 kDa), and HMGB1 (25 kDa). C: Levels of secreted HMGB1 were determined in culture medium by enzyme-linked immunosorbent assay (ELISA) (n = 5). Values are shown as means + SD, *P < 0.05 vs. LG/CTL group. (A high-quality color digital representation of this figure is available in the online issue.)

RAGE (Receptor for Advanced Glycation End Products) はパターン認識受容体であり、正常状態では免疫系の恒常性に寄与している。しかし、糖尿病により恒常性が崩壊すると、 RAGE や RAGE リガンドの発現上昇を伴って、慢性炎症を促進し、糖尿病合併症を誘発する。これまでに筆者らは、高血糖により誘導された活性酸素種 (ROS) が糖尿病関連の疾病を誘発する経路の一つとして、細胞内での AGEs 産生の増加を見出している。しかしながら、 ROS が RAGE および RAGE リガンドに与える影響については検討されていなかった。そこで本論文では、高血糖により誘導された ROS が RAGE および RAGE リガンドの発現に与える影響を評価した。

【背景と目的】

【略語】

AGE :RAGE :UCP1 :SOD2 :GLO1 :

MG :MnTBAP :

CM-H2DCFDA :

Advanced Glycation End ProductsReceptor for AGEUncoupling Protein 1Superoxide Dismutase 2Glyoxalase 1MethylglyoxalMn( )tetrakis(4-benzoic acid)porphyrin chlorideⅢchloromethyl-dichlorodihydrofluorescein diacetate

HGRAGE

RAGE Ligand

UCP1SOD2GLO1

High Glucose

ROS

GAPDH

MG

AGEs

MG-mSin3A

MG

High Glucose

mSin3A

SP3 Angiopoietin2

【 筆 者 ら の 以 前 の 研究】

FIG. 2. Overexpression of UCP1, SOD2, and GLO1 prevents hyperglycemia-induced ROS generation and methylglyoxal formation. Primary HAECs were infected with UCP1, SOD2, GLO1, or empty control adenovirus (CTL). After incubation with either low glucose (LG) (5 mmol/l) or high glucose (HG) (30 mmol/l) for 1 or 5 days, the cells were used for analysis. A: ROS formation on day 5. B: Methylglyoxal-modified protein on day 5. C: Representative blots for B. D: GLO1 activity on day 1. E: GLO1 protein on day 1. F: Representative blots for E. G: GLO1 activity on day 5. H: GLO1 protein level on day 5. I: Representative blots for H. n = 4. *P < 0.05 vs. LG/CTL group. Values are shown as means + SD. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 3. GLO1 knockdown duplicates the effect of hyperglycemia on expression of RAGE, S100 calgranulins, and HMGB1. Conditionally immortalized HAECs were infected with either shGLO1 or nontargeting control (CTL) lentivirus and then incubated in either low glucose (LG) or high glucose (HG) for 5 days. A: mRNA levels were determined by real-time PCR (n = 3). B: Protein levels were determine by Western blotting (n = 4). Protein sizes were evaluated and confirmed by standard protein markers, and their sizes were as follows: RAGE (46 kDa), S100A8 (11 kDa), and HMGB1 (25 kDa). C: Intracellular methylglyoxal-modified proteins were quantitated by Western blotting (n = 4). D: Representative blots for C. Values are shown as means + SD. *P < 0.05 vs. CTL group. (A high-quality color digital representation of this figure is available in the online issue.)

HG

ROSAGEs

UCP1SOD2GLO1

GLO1

GLO1 knock downRAGE

RAGE LigandAGEs

FIG. 4 Hyperglycemia-induced methylglyoxal increases binding of NFκB to the RAGE promoter and binding of AP-1 to the S100A8, S10012, and HMGB1 promoters. A: Conditionally immortalized HAECs were treated with low glucose (LG), high glucose (HG), or HG after transfection with either scrambled oligonucleotides (LG and HG) or siRNA for p65 (HG/siP65) for 5 days, and the RAGE mRNA level was determined by qPCR (n = 3). B: Primary HAECs were treated with LG, HG, or HG after infections with either nontarget control or GLO1 adenovirus (HG/GLO1) for 5 days. Chromatin immunoprecipitation was performed using the p65 antibody, and the RAGE promoter was amplified by qPCR (n = 4). C: Conditionally immortalized HAECs were treated with LG, HG, or HG after transfection with either scrambled oligonucleotides or siRNA for AP-1 (c-Jun) (HG/siAP-1) for 5 days, and mRNA levels of S100A8, S10012, and HMGB1 were determined by qPCR (n = 3). D: Primary HAECs were treated with

LG, HG, or HG after infections with either nontarget control or GLO1 adenovirus (HG/GLO1) for 5 days. Chromatin immunoprecipitation was performed using c-Jun antibody, and the S100A8, 100A12, and HMGB1 promoters were amplified by qPCR (n = 4). Values are shown as means + SD. *P < 0.05 vs. LG group. (A high-quality color digital representation of this figure is available in the online issue.)

FIG. 5. Treatment of diabetic mice with MnTBAP normalizes increased expression of RAGE, S100 calgranulins, and HMGB1. A: Aortic endothelial cells (AEC) or smooth muscle cells (SMC) were isolated from either wild-type (WT) mice, STZ-induced diabetic mice (STZ), or STZ-diabetic mice treated with MnTBAP (STZ/MnTBAP), using laser capture microdissection. mRNA levels for vWF (A) and α-actin (B) were determined by qPCR (n = 3, *P < 0.05 vs. AEC group). C: 3-Nitrotyrosine content of aortic cells from the above mice was determined by Western blot (n = 4, *P < 0.05 vs. CTL group). D: Representative blots for C. E: Methylglyoxal-modified protein content of aortic cells from the above mice was determined by Western blot (n = 4, *P < 0.05 vs. CTL group). F: Representative blots for E. G: Aortic endothelial cells were used for analysis of gene expression by qPCR (n = 3, *P < 0.05 vs. WT group). Values are shown as means + SD. (A high-quality color digital representation of this figure is available in the online issue.)

GLO-1

HG NF-κB RAGE

HG AP-1 RAGE Ligand

HG

RAGERAGE Ligand

ROSAGEs

MnTBAP

【 ま とめ】

高血糖により誘導される ROS が

RAGE内因性 RAGE リガンドの発現を促進した。

GLO1 がこのメカニズムに対して、抑制的に働くことが示された。

ROS GLO1

NF-κB RAGE

AP-1 RAGE Ligand

MG

High Glucose

MG-AGEs